Figure 4. H 2 R inhibits OL... | Rockefeller University Press

Figure 4.

H2R inhibits OL differentiation by binding with axin2 to up-regulate the Wnt/β-catenin signaling pathway.(a) Representative co-immunoprecipitation results showing the interaction of H2R with Axin2 following OGD/reperfusion (bands of GFP are located at ~95 kD). (b–d) Western blot analysis showing the effect of H2R overexpression (AAV-Hrh2) or knockdown (AAV-sh-Hrh2) on β-catenin, p-GSK3β, and GSK3β expression in OGD-treated oli-neu cells. (e) Western blot analysis showing the effect of Flag-Hrh2 plasmid cotransfection with GFP-Axin2 plasmid on β-catenin expression compared with the Flag-Hrh2 single transfection in OGD-treated oli-neu cells. (f) Western blot analysis showing the effect of small interfering (si)-Hrh2 cotransfection with si-Axin2 on β-catenin expression compared with the si-Hrh2 single transfection in OGD-treated oli-neu cells. (g and h) Immunocytochemical visualization and quantification of primary O4+ multipolar differentiating OLs that received the transfection of AAV-Hrh2 and Axin2 plasmid (g) or transfection of AAV-sh-Hrh2 and si-Axin2 (h) and exposure to OGD/reperfusion. n = 4–7 from at least three independent experiments. Scale bar, 50 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001. co-IP, co-immunoprecipitation; CON, control; Veh, vehicle.

H₂R inhibits OL differentiation by binding with axin2 to up-regulate the Wnt/β-catenin signaling pathway. (a) Representative co-immunoprecipitation results showing the interaction of H₂R with Axin2 following OGD/reperfusion (bands of GFP are located at ~95 kD). (b–d) Western blot analysis showing the effect of H₂R overexpression (AAV-Hrh2) or knockdown (AAV-sh-Hrh2) on β-catenin, p-GSK3β, and GSK3β expression in OGD-treated oli-neu cells. (e) Western blot analysis showing the effect of Flag-Hrh2 plasmid cotransfection with GFP-Axin2 plasmid on β-catenin expression compared with the Flag-Hrh2 single transfection in OGD-treated oli-neu cells. (f) Western blot analysis showing the effect of small interfering (si)-Hrh2 cotransfection with si-Axin2 on β-catenin expression compared with the si-Hrh2 single transfection in OGD-treated oli-neu cells. (g and h) Immunocytochemical visualization and quantification of primary O4⁺ multipolar differentiating OLs that received the transfection of AAV-Hrh2 and Axin2 plasmid (g) or transfection of AAV-sh-Hrh2 and si-Axin2 (h) and exposure to OGD/reperfusion. n = 4–7 from at least three independent experiments. Scale bar, 50 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001. co-IP, co-immunoprecipitation; CON, control; Veh, vehicle.